D (Fig 3F). To decide whether the truncations decreased the activity toward phospho-ERK through recognition

D (Fig 3F). To decide whether the truncations decreased the activity toward phospho-ERK through recognition of the ERK activation loop sequence, we measured the STEP truncation activity toward the ERK pT202pY204 phospho-peptide. All truncations had kcat/Km ratios for this phospho-ERK TXB2 Purity & Documentation peptide that were comparable towards the wild-type phosphatase, suggesting that these truncations do not have an effect on STEP activity via a loss of phospho-peptide sequence recognition. As a result, KIM, the N-terminal portion of KIS, plus the C-terminal a part of KIS are necessary for ERK dephosphorylation by STEP. These motifs contribute to dephosphorylation via protein-protein interactions in lieu of by affecting the intrinsic activity of STEP or its recognition with the ERK phospho-peptide sequence. Residues from the STEP KIM area responsible for efficient phospho-ERK dephosphorylation In addition to STEP, a minimum of two recognized ERK tyrosine phosphatases (HePTP and PTP-SL) and most dual-specificity MAP kinase phosphatases possess a KIM that mediates their interactions with ERK(Francis et al. 2011a) (Zhou et al. 2002). Biochemical and structural experiments have revealed that two conserved basic residues followed by the hydrophobic A-X-B motif mediate ERK-phosphatase interactions through STEP binding to the CD internet site as well as a hydrophobic groove positioned on the ERK surface, respectively (Fig 4A) (Liu et al. 2006, Piserchio et al. 2012b, Huang et al. 2004, Zuniga et al. 1999). Depending on our previous crystallographic function on the ERK-MKP3 interaction, we also generated a structural model of ERK in complex with STEP-KIM to facilitate our mutagenesis design and style (Fig 4C, solutions in supplemental components). To get insight into how KIM mediates the dephosphorylation of ERK by STEP, we initially mutated the conserved simple residue R242 or R243 as well as the hydrophobic residue L249 or L251 and monitored the effects of those mutants on STEP catalysis. Similar towards the STEPKIM SIRT2 site deletion, these mutations did not influence STEP activity toward pNPP or the phosphopeptide derived in the ERK activation loop (Fig 4B). Having said that, the mutation of eitherJ Neurochem. Author manuscript; readily available in PMC 2015 January 01.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLi et al.PageR242A or R243A decreased the kcat/Km ratio from the reaction toward the phospho-ERK protein by 4- or 6-fold, respectively (Fig 4B). These final results suggest that these mutations mainly impaired the binding of STEP to ERK. We subsequent examined the effects of mutations inside the conserved hydrophobic A-X-B motif of STEP. Our structural model predicted that STEP L249 sits in a pocket defined by H142, Y145 and F146, of ERK, whereas STEP L251 is positioned inside the hydrophobic pocket defined by ERK L132 and L173 (Fig 4C). Mutation of L249A or L251A decreased the kcat/Km for phospho-ERK by 2.5-fold or 7-fold, respectively (Fig 4B). Therefore, we conclude that each conserved hydrophobic residues inside the A-X-B motif along with the arginine situated in KIM are vital for efficient ERK dephosphorylation by STEP. S245, situated inside the STEP KIM, is definitely an vital regulatory internet site inside the dephosphorylation of phospho-ERK by STEP It’s worth noting that STEP activity is downregulated by the phosphorylation of Ser245 in KIM, which can be mediated by the activation of D1 dopamine receptor stimulated by psychostimulant drugs (Valjent et al. 2005, Paul et al. 2000). Conversely, NMDA receptor activation results in STEP dephosphorylation at Ser245 by calcineurin, activating STEP.